Hydrolytic Glycosidic Bond Cleavage in RNA Nucleosides: Effects of

Nov 23, 2016 - Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta T1K 3M4, Canada...
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Hydrolytic Glycosidic Bond Cleavage in RNA Nucleosides: Effects of the 2′-Hydroxy Group and Acid−Base Catalysis Stefan A. P. Lenz, Johnathan D. Kohout, and Stacey D. Wetmore* Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta T1K 3M4, Canada S Supporting Information *

ABSTRACT: Despite the inherent stability of glycosidic linkages in nucleic acids that connect the nucleobases to sugar−phosphate backbones, cleavage of these bonds is often essential for organism survival. The current study uses DFT (B3LYP) to provide a fundamental understanding of the hydrolytic deglycosylation of the natural RNA nucleosides (A, C, G, and U), offers a comparison to DNA hydrolysis, and examines the effects of acid, base, or simultaneous acid−base catalysis on RNA deglycosylation. By initially examining HCOO−···H2O mediated deglycosylation, the barriers for RNA hydrolysis were determined to be 30−38 kJ mol−1 higher than the corresponding DNA barriers, indicating that the 2′-OH group stabilizes the glycosidic bond. Although the presence of HCOO− as the base (i.e., to activate the water nucleophile) reduces the barrier for uncatalyzed RNA hydrolysis (i.e., unactivated H2O nucleophile) by ∼15−20 kJ mol−1, the extreme of base catalysis as modeled using a fully deprotonated water molecule (i.e., OH− nucleophile) decreases the uncatalyzed barriers by up to 65 kJ mol−1. Acid catalysis was subsequently examined by selectively protonating the hydrogen-bond acceptor sites of the RNA nucleobases, which results in an up to ∼80 kJ mol−1 barrier reduction relative to the corresponding uncatalyzed pathway. Interestingly, the nucleobase proton acceptor sites that result in the greatest barrier reductions match sites typically targeted in enzyme-catalyzed reactions. Nevertheless, simultaneous acid and base catalysis is the most beneficial way to enhance the reactivity of the glycosidic bonds in RNA, with the individual effects of each catalytic approach being weakened, additive, or synergistic depending on the strength of the base (i.e., degree of water nucleophile activation), the nucleobase, and the hydrogenbonding acceptor site on the nucleobase. Together, the current contribution provides a greater understanding of the reactivity of the glycosidic bond in natural RNA nucleosides, and has fundamental implications for the function of RNA-targeting enzymes.

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INTRODUCTION DNA and RNA are stable nucleic acid polymers that participate in a wide variety of cellular functions, including information storage and protein synthesis.1−8 Cleavage of the glycosidic linkage between a nucleobase and the sugar−phosphate backbone occurs during many cellular pathways. For example, deglycosylation is a step in base excision repair of damaged DNA initiated by DNA glycosylases,2−4 ribosome inactivation catalyzed by RNA glycosidases,5,6 and purine or pyrimidine salvage facilitated by RNA nucleoside hydrolases (NH).7,8 Furthermore, these pathways are often essential for organism survival. For example, breakdown of the base excision repair pathway has been linked to increased incidences of many different types of cancer2 and ricin-A enzymes that catalyze ribosome inactivation have been identified as potent poisons.6,9 Similarly, purine or pyrimidine salvage is an important biological process in organisms including parasitic protozoa, which cause malaria,10 African and American trypansomiasis,7,11,12 and leishmaniasis.12,13 Since the glycosidic bonds in nucleic acids are inherently stable, a number of experimental studies have focused on understanding how glycosidic bond cleavage is catalyzed by critical cellular machinery including DNA glycosylases,14−18 RNA glycosidases,9,19−21 and RNA NH.22−25 The associated deglycosylation reactions entail attack by a nucleophile (water © XXXX American Chemical Society

or amine) at the anomeric (C1′) carbon, and nucleobase departure. These enzymes have been proposed to utilize diverse catalytic strategies including the following: (1) stabilization of the negative charge developing on the nucleobase via protonation,14,15 hydrogen bonding,9,16,20,21 or π−π interactions;17,22 (2) stabilization of the positive charge developing on the sugar group via strategic positioning of basic residues (Asp/ Glu);23,24 and (3) activation of the nucleophile that displaces the nucleobase.18,19,25 While it is clear that each of these strategies can reduce an otherwise prohibitive deglycosylation barrier, the catalytic contribution afforded by each avenue may be dependent on the substrate targeted (e.g., damaged versus undamaged nucleobase, deoxyribose versus ribose sugar, nucleoside versus nucleotide) and the enzyme involved. For example, the DNA glycosylase MutY and RNA hydrolase IUNH may facilitate deglycosylation via nucleobase protonation,14,15,23 while DNA-targeting UDG and RNA-targeting CUNH may achieve catalysis via hydrogen bonding between the departing nucleobase and catalytic active site residues.16,26,27 On the other hand, AAG (DNA glycosylase) and IAG-NH (RNA NH) have been proposed to stabilize the leaving group Received: September 22, 2016 Revised: November 10, 2016 Published: November 23, 2016 A

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The Journal of Physical Chemistry B via π−π interactions between the departing nucleobase and aromatic amino acids.17,22,28 To understand the driving force for an enzyme to take advantage of different catalytic approaches, the relative effect of each strategy or combination of strategies on the reaction must be determined. With the goal of elucidating the general impact of various factors on DNA deglycosylation, our group previously used carefully designed computational models that are not specific to a particular enzyme.29−37 Initially, the concerted (SN2) deglycosylation of the uracil nucleoside (a common type of DNA damage) was considered, including the effects of hydrogen-bonding interactions between small molecules (hydrogen fluoride, water, or ammonia) and different acceptor sites of the nucleobase.29 In a follow-up study, the relative stability of the glycosidic bond in the natural DNA nucleosides was determined, with and without hydrogen-bonding interactions between small molecules and the nucleobases.30,34,35 The computational model was also scrutinized by examining whether the inclusion of a phosphate moiety changes the reaction pathway (i.e., nucleoside versus nucleotide models), and how the inclusion of implicit solvent within the optimization routine affects the structures of stationary points and reaction energetics.35 Furthermore, the effects of different nucleophiles (i.e., amine, proline, or water),33,38,39 mechanisms (i.e., SN1 versus SN2),31,32,37 and damaged (e.g., 8-oxoguanine and thymine glycol)31,33,36,37 or activated (i.e., via hydrogen bonding, protonation, or π−π interactions)34,37 DNA nucleobases were considered. These works were augmented by other contributions in the literature that used small models to investigate the impact of nucleobase oxidation40−42 and/or activation (i.e., protonation or metal ions)40−44 on DNA deglycosylation, as well as larger protein−DNA models to focus on specific enzymes (i.e., DNA glycosylases).45−59 Despite detailed studies on DNA deglycosylation, little is known about the corresponding reaction in RNA. Although computational work has considered select aspects of specific enzyme-catalyzed reactions,28,60−69 such studies do not reveal the intrinsic stability of the glycosidic bond in RNA or comprehensively isolate ways the associated barrier can be reduced across all natural RNA nucleosides. Thus, while enzymes such as RNA glycosidases and nucleoside hydrolases may use at least some of the catalytic strategies outlined above to facilitate deglycosylation, the individual contributions of each approach are currently unclear. Furthermore, while an experimental study has reported greater stability of the glycosidic bond in RNA versus DNA purines under acidic conditions (pH 1), there is currently no comparable data for the pyrimidines nor a comparison of acid and base catalysis.70 Therefore, the intrinsic effect of 2′-OH on the stability of the glycosidic bond is unknown. Since work on DNA has shown that small model computational studies can provide important information about nucleic acid deglycosylation and thereby expand our fundamental understanding of the corresponding enzymatic reactions,29−37 the present work uses computational methods to comprehensively study the hydrolysis of the four canonical RNA nucleosides (A, C, G, and U, Figure 1). Specifically, we first examine deglycosylation using formate activated water (HCOO−···H2O) as the nucleophile, since formate contains the functionality of the catalytic Asp/Glu residue present in the active site of many nucleic acid-targeting enzymes (e.g., DNA glycosylases,14−18,71 RNA glycosidases,6,9,19−21 and RNA NH).22−25,72 Using this model, we compare RNA and DNA

Figure 1. Structure and chemical numbering of the canonical RNA (R = ribose) and DNA (R = deoxyribose) nucleosides.

hydrolysis to determine the impact of 2′-OH on both the structures of the stationary points and the reaction energetics. Subsequently, to quantify the effects of base and/or acid catalysis, we characterize uncatalyzed RNA nucleoside hydrolysis using a single (unactivated) water molecule as the nucleophile. Since glycosidic bond cleavage barriers have been shown to be dependent on the species accepting a proton from the nucleophilic water,29 stronger base catalysis than provided by formate is then examined using hydroxide (OH−) as the nucleophile, which represents the extreme of full water activation. To solely consider acid catalysis, we sequentially protonate each hydrogen-bond acceptor site in the RNA nucleobases using a model containing an unactivated H2O nucleophile. Finally, to determine whether the effects of base and acid catalysis are additive, we examine the excision of protonated nucleobases facilitated by activated water (i.e., the HCOO−···H2O or OH− nucleophile). The current study uncovers the inherent stability of the glycosidic bond in the natural RNA nucleosides, as well as the stability relative to the corresponding DNA analogues. Furthermore, the effects of the reaction environment (i.e., neutral versus basic versus acidic conditions) on the deglycosylation reaction are also revealed. When combined with previous computational and experimental studies, the current work yields valuable insights into strategies that can be exploited by enzymes that target RNA and provides a starting point for developing larger computational models to study enzyme-catalyzed RNA deglycosylation. Furthermore, the present work provides structural insights that can be exploited in the future development of drugs that combat disease by inhibiting RNA-targeting enzymes (e.g., block RNA NH to treat trypansomatid diseases,7,11,12 such as American and African trypansomiasis, and leshmaniasis).

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COMPUTATIONAL DETAILS Due to the insights acquired from previous computational studies of DNA deglycosylation,9,14−25 an analogous methodology was used in the present work to gain a fundamental understanding of RNA hydrolysis. This approach will also afford an accurate comparison of RNA and DNA deglycosylation, which was one of the driving forces behind the present study. Specifically, our computational models were built by adding a 2′-hydroxy group to the previously reported35 DNA nucleoside models that include a HCOO−···H2O nucleophile (Figure 2a). The formate in the previous DNA models B

DOI: 10.1021/acs.jpcb.6b09620 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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calculated using small models with methyl and phosphate caps are minimal for DNA hydrolysis,35 and therefore, our models are also applicable to enzymes that bind to RNA polymers rather than free nucleosides or nucleotides. Using the model described above (Figure 2a), a hydrogen bond forms between the HCOO− anion and 2′-OH (Figure S1). This interaction is unlikely to occur in an enzymecatalyzed mechanism, since the 2′-hydroxy group typically interacts with other active site residues. For example, in RNA NH, the 2′-OH oxygen atom is coordinated to a Ca2+ ion, while the hydrogen atom is typically occupied by interactions with either another Asp/Glu (that does not activate the nucleophile) or a backbone carbonyl of a neighboring residue.26,72 In the case of the ricin-A chain, the 2′-hydroxy group of the bound adenosine monophosphate substrate is in position to donate a hydrogen bond to an active site tyrosine residue.75 Therefore, to occupy 2′-OH in our models, an explicit water molecule was introduced that bridges between O2′ and O3′ (Figure 2b). This choice is also chemically reasonable, since a water molecule could occupy this position under experimental conditions used to study the nonenzymatic hydrolysis reactions. Furthermore, since a previous study from our group determined that the inclusion of solvent in the optimization routine yields structures and barriers in better agreement with experiment,35 we used the IEF-PCM implicit solvent model with a dielectric constant of water (78.3553) to allow closer comparisons to the experimental data obtained for the hydrolysis reactions. To examine the effect of base and/or acid catalysis on the reaction barrier, we implemented several small models. First, to provide a point of reference to determine catalytic effects, we characterized the uncatalyzed pathways using an unactivated

Figure 2. Models used in the present study to examine RNA nucleoside deglycosylation.

represents the carboxylate side chain of Asp/Glu in the active site of many DNA glycosylases that activates the water nucleophile.14−18 This nucleophile model was retained in the present work due to the similar role Asp/Glu may play in RNA NH and glycosidase-catalyzed reactions.9,19−25 Additionally, we maintained the methyl caps on the 3′ and 5′ oxygen atoms instead of hydrogen atoms in order to eliminate hydrogen bonding between these groups and the departing nucleobase that is unlikely to occur in enzymatic systems. For example, the 5′- and 3′-hydroxy groups interact with active site residues when RNA substrates are bound to IAG-NH and CU-NH.24,73 Furthermore, although other RNA-targeting enzymes (such as the ricin-A chain) bind to rRNA rather than free nucleosides,74,75 the differences between structures and barriers

Figure 3. Selected IEF-PCM-B3LYP/6-31G(d) bond lengths (Å) and angles (degrees in parentheses) in reactant (RC), transition state (TS), and product (PC) complexes for the deglycosylation of adenine (A), cytosine (C), guanine (G), and uracil (U) containing (a) RNA and (b) DNA nucleosides facilitated by the HCOO−···H2O nucleophile. C

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The Journal of Physical Chemistry B water nucleophile (Figure 2c). Subsequently, to examine the effect of increasing nucleophile activation, we examined pathways catalyzed by a fully activated water (i.e., OH− nucleophile, Figure 2d). Finally, to evaluate the effect of acid catalysis or the combined effect of acid−base catalysis, the hydrogen-bond acceptor sites on the nucleobases (Figure 1) were sequentially protonated in models containing an unactivated or activated (HCOO−···H2O or OH−) water nucleophile. In each model considered, SN2 transition states for RNA nucleoside deglycosylation were optimized at the B3LYP/631G(d) level in implicit water (IEF-PCM) using Gaussian 09 default convergence criteria. The choice to solely examine SN2 mechanisms stems from the small barrier difference previously reported for SN1 and SN2 deglycosylation in DNA nucleosides,37 and this choice allows direct comparisons to our corresponding DNA hydrolysis studies.35 Subsequently, intrinsic reaction coordinate (IRC) calculations were performed to confirm the connectivity of the transition state (TS) to the corresponding reactant (RC) and product (PC) complexes. The RC and PC were fully optimized to minima, and the identity of each stationary point (RC, TS, and PC) was confirmed through frequency calculations. Scaled (0.9806)76 zero-point and thermal corrections were obtained from harmonic frequencies performed with IEF-PCM-B3LYP/631G(d) and applied to SMD-B3LYP-D3/6-311+G(2df,2p) single-point energies to yield the reported Gibbs energies. The SMD implicit solvation model was implemented, since this methodology has been shown to provide more accurate solvated Gibbs energies.77 All calculations were performed with the Gaussian 09 program suite (revision A.02, C.01, or D.01).78

the site of reaction, the reaction angle increases to 160°. The nucleobase is fully dissociated in the PC, with a glycosidic bond distance of ∼3.500−4.000 Å. Hydrogen bonding is observed between the departed nucleobase and the sugar−nucleophile moiety, which stabilizes the negative charge on the nucleobase (Figure 3a). These hydrogen bonds change depending on the identity of the nucleobase, with N3 of the purines or O2 of the pyrimidines interacting with H1′. Full proton transfer from the water nucleophile to HCOO− is observed for each reaction, with the Owat−Hwat···OHCOO− distance shortening from ∼1.800 Å in the RC to 1.000 Å in the PC (Tables S1−S4). The reaction barriers and energies for the hydrolytic deglycosylation of the RNA nucleosides range from 152.9 to 185.6 kJ mol−1 (Table 1). U has the least stable glycosidic bond

RESULTS AND DISCUSSION Hydrolysis Facilitated by the HCOO−···H2O Nucleophile. RNA Deglycosylation. As discussed in the Computational Details, HCOO− in our initial model (Figure 2b) represents the side chain of an Asp or Glu that is proposed to be the general base in reactions catalyzed by RNA-targeting nucleoside hydrolases8,22−25 and glycosidases.6,9,19−21 Structures and key geometric parameters along the HCOO−···H2O hydrolysis pathway of RNA nucleosides are shown in Figure 3a, while detailed geometric data is provided in Tables S1−S4. Regardless of the RNA nucleoside considered, the nucleophilic (C1′−Owat) distance shortens, while the glycosidic bond (C1′− N1 for pyrimidines or C1′−N9 for purines) distance elongates until the nucleobase is fully dissociated from the sugar moiety, and a covalent bond is formed between C1′ and the nucleophile. The hydrogen-bond distance between 2′-OH and the bridging water molecule (O2′−H2′···Obw) is ∼1.800 Å (∠(H2′O2′Obw): ∼165°) throughout the reaction (Figure 3a), which indicates that inclusion of this bridging water will not significantly affect the reported barriers and reaction energies (rxn). In the RC, the nucleophile is ∼3.300−3.400 Å away from C1′ and the reaction angle [∠(OwatC1′N1/N9)] is near 130° for each canonical RNA nucleoside (Figure 3a). The water nucleophile forms hydrogen bonds with both O4′ and HCOO−, while HCOO− also hydrogen bonds with H4′ of the sugar (Tables S1−S4). Therefore, the water nucleophile is well positioned for SN2 attack. The glycosidic bond length in the TS elongates to ∼2.400−2.500 Å, while the nucleophilic distance shortens to ∼2.200 Å. As the nucleophile approaches

(152.9 kJ mol−1) and C the most stable glycosidic bond (185.6 kJ mol−1), while deglycosylation of the purines results in approximately equivalent barrier heights (within 6 kJ mol−1, Table 1). This correlates with the hydrolysis of U having the earliest TS (C1′−N1, 2.426 Å; C1′−Owat, 2.189 Å) and C being a poorer leaving group with the latest TS (C1′−N1, 2.477 Å; C1′−Owat, 2.216 Å), as well as the hydrolysis of the purines resulting in nearly identical coordinates for the bond forming and breaking events. These results are also supported by the greater calculated N1 acidity for the U compared to C nucleobase and the near equivalent N9 acidities of the A and G nucleobases.37 Although the reactions are endothermic (by 55.7−98.1 kJ mol−1), the reaction energies are substantially lower than the barrier heights (by ∼100 kJ mol−1), which at least in part reflects stability provided by the hydrogen bonds between the nucleobase and sugar−nucleophile moiety in the PC. Comparison of RNA and DNA Deglycosylation. In order to determine the effect of the 2′-hydroxy group on nucleic acid hydrolysis, we compare RNA and DNA nucleoside deglycoyslation facilitated by the HCOO−···H2O nucleophile. To the best of our knowledge, no direct comparisons of the hydrolysis reactions for all canonical RNA and DNA nucleosides has been made to date. Stationary points along the SN2 reaction pathways of DNA (A, C, G, and U) deglycosylation catalyzed by HCOO−···H2O are shown in Figure 3b.35 Although not a canonical DNA nucleobase, U is a common type of DNA oxidation damage.2 Since a previous study found little difference in the reaction energetics ( A = G > U, which further highlights differences in the intrinsic stability of the glycosidic bond across the RNA nucleosides. Additionally, the reaction energies are only ∼10 kJ mol−1 more stable than the TS for uncatalyzed hydrolysis, indicating that the PC are transient and re-emphasizing the necessity of catalysis. This contrasts the ∼100 kJ mol−1 in stability of the HCOO−···H2O PC relative to the corresponding TS. Base Catalysis. Since water nucleophile activation by formate affects the structures and relative energies of stationary points along the uncatalyzed reaction pathway, deglycosylation catalyzed by OH− is now considered to understand the extreme effect of full water activation (full base catalysis). We note that our pathways do not include the energetic cost associated with deprotonating a water molecule, since we are primarily interested in the extreme effect of water activation in the presence of a strong base. Indeed, a previous study compared deglycosylation by water activated using small molecules with a range in basicities (e.g., halogens, HCOO−, and CN−) and hydrolysis by OH−, and determined that OH− yields the lowest deglycosylation barriers.29 In the OH− catalyzed pathways, the RC nucleophilic distances are ∼2.900−3.100 Å (Figure 5 and Tables S10−S13), which is 0.600−0.700 Å shorter than for the corresponding uncatalyzed reactions. Additionally, the glycosidic bond length in the TS for the OH− catalyzed pathways is ∼1.900−2.000 Å, which represents a 0.500−0.600 Å decrease relative to the uncatalyzed pathways. The OH− TS also have significantly shorter glycosidic bonds than the HCOO−···H2O catalyzed pathways (by ∼0.400 Å). The earlier TS for the OH− catalyzed reaction likely results from the increased nucleophilic power of fully activated water over the unactivated water nucleophile. Furthermore, the negative charge of OH− helps stabilize the positive charge developing on the sugar in the TS. The fact that enhancement in nucleophilic power results in earlier TS has been previously reported for DNA hydrolysis.30,35 In the PC, the C1′−N1 (C or U) or C1′−N9 (A or G) distance is ∼3.500 Å, which is 0.500 Å shorter than observed for the HCOO−···H2O catalyzed PC (Figures 3 and 5). Regardless, the O2′−H2′···O bw hydrogen bond is maintained, and does not significantly fluctuate along the reaction pathway (Figure 5 and Tables S10−S13). The structural differences between the OH− catalyzed and uncatalyzed hydrolysis pathways result in ∼40−60 kJ mol−1 lower barriers when OH− is the nucleophile (Δbase(OH−), Table 2). The energetic difference is most pronounced for C (62.5 kJ mol−1) and least pronounced for A (43.4 kJ mol−1),

Figure 5. Selected IEF-PCM-B3LYP/6-31G(d) bond lengths (Å) and angles (degrees in parentheses) in reactant (RC), transition state (TS), and product (PC) complexes for the deglycosylation of adenine (A), cytosine (C), guanine (G), and uracil (U) containing RNA nucleosides facilitated by the OH− nucleophile.

while G (48.8 kJ mol−1) and U (51.4 kJ mol−1) exhibit a similar nucleophile dependence as A. Due to these differences, the relative stability of the glycosidic bond among the canonical RNA nucleosides changes depending on the nucleophile. Specifically, the stability decreases as G = A > C > U when OH− is the nucleophile but as C > G > A > U when unactivated H2O or HCOO−···H2O is the nucleophile. The reaction energies also change significantly with the level of nucleophile activation. Specifically, the PC are more stable than the RC in the OH− pathways (reaction energies ∼ −60 to −90 kJ mol−1, Table 2), while the H2O (reaction energies ∼155−190 kJ mol−1, Table 2) and HCOO−···H2O (reaction energies ∼55− 100 kJ mol−1, Table 2) mediated pathways are endothermic. The deviations in the reaction pathways and energetics with the nucleophile highlight the importance of nucleophile activation in enzyme-catalyzed RNA deglycosylation, and the possible role of the nucleophile as a rate determinant. Notably, the catalytic rates of RNA nucleoside hydrolases deviate significantly depending on their substrate specificity (i.e., purine specific, pyrimidine specific, or purine−pyrimidine nonspecific).23,81−84 Although the difference in activity may be at least in part attributed to the varied reactivity of the nucleosides, our work shows that the variation in the barrier with nucleoside (up to ∼20 kJ mol−1) is less than the variation in the barrier with nucleophile (up to ∼60 kJ mol−1, Table 2). Therefore, the present work suggests the deviation in catalytic rates exhibited by RNA-targeting enzymes may be a consequence of the position and orientation of the general base, which may affect nucleophile activation and charge stabilization during (for SN2) or post (for SN1) nucleobase excision. Nevertheless, despite significant barrier reductions for the fully activated nucleophile (OH−) over the uncatalyzed deglycosylation pathways, the barriers remain high (up to ∼145 kJ mol−1, Table 1), and therefore, enzymes must use other strategies to further catalyze RNA hydrolysis. G

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The Journal of Physical Chemistry B Effects of Acid Catalysis. Direct protonation of the nucleobase has been proposed to facilitate RNA deglycosylation in a number of enzyme-catalyzed reactions. For example, O2 protonation of C by E. coli inosine-uridine nucleoside hydrolase (RihC) has been proposed to be an essential step in the catalytic deglycosylation mechanism,85 while N7 protonation of the purine has been proposed to play a role in the catalytic deglycosylation facilitated by Crithidia fasciculate IU-NH.86,87 In the current study, the effect of acid catalysis is initially considered by investigating full protonation at each possible nucleobase acceptor site (Figure 1) for the model with unactivated water as the nucleophile (Figure 2b). Protonation rather than hydrogen bonding is considered, since protonation will likely result in the greatest reductions in the hydrolysis barriers. Indeed, when HCOO−···H2O is the nucleophile, protonation of the uracil DNA nucleoside37 results in at least a 20 kJ mol−1 reduction in the deglycosylation barrier calculated when a highly polar acid (HF) hydrogen bonds with U.35 Structures for the deglycosylation pathways corresponding to the protonation site that leads to the lowest barrier for each RNA nucleoside are shown in Figure 6, and will be discussed as

as C−O2H(N3)). The reactant structures for acid catalysis (Figure 6) resemble those for the (uncatalyzed) H2O-mediated pathways (Figure 5), with H2O positioned for SN2 attack at C1′. The nucleophilic distance is ∼3.200−3.400 Å, which is shorter than observed for the uncatalyzed pathways (∼3.400− 3.500 Å). As with the uncatalyzed pathways, the nucleophile donates a hydrogen bond to the 2′-hydroxyl group, which donates a hydrogen bond to the bridging water molecule, and these contacts are maintained throughout the reaction pathways. In the TS, the glycosidic bond distance falls between 2.500 and 2.600 Å, and the nucleophilic distance, between ∼2.200 and 2.500 Å, depending on the identity of the nucleobase. The hydrogen bonds between the water nucleophile and 2′-OH, as well as the bridging water molecule and 2′-OH, tighten by up to 0.100 Å in the TS compared to the RC, which helps stabilize the positive charge developing on the sugar (Figure 6 and Tables S6−S9). Similarly, the reaction angle increases from ∼125° in the RC to ∼150° in the TS. The PC contain fully formed bonds between the water nucleophile and ribose, while the nucleobase has departed, falling 3.100− 3.500 Å from C1′ as discussed for the uncatalyzed pathways. The Owat−Hwat···O2′ hydrogen-bond distance decreases by ∼0.200 Å from the TS to the PC, indicating partial proton transfer to the 2′-hydroxyl group. The hydrogen bond between 2′-OH and the bridging water shortens to ∼1.650 Å in the PC, which stabilizes the cationic charge forming on ribose as observed for the H2O-mediated pathways. The barriers for all of the acid-catalyzed pathways range from 89.7 to 162.4 kJ mol−1 (Table 2), and acid catalysis results in a 23.5−82.5 kJ mol−1 reduction in the reaction barrier over the corresponding uncatalyzed reactions. U−O2H(N3) has the smallest barrier (89.7 kJ mol−1), which is consistent with the considerably longer nucleophilic distance in the U−O2H(N3) TS (2.538 Å) compared to the other TS (∼2.2−2.3 Å; Figure 6). Indeed, protonation of O2 results in the largest barrier reduction (∼80 kJ mol−1) for both pyrimidines (Δacid, Table 2). Although the orientation of the proton on O2 greatly affects the barrier for U (i.e., a ∼ 35 kJ mol−1 dependence on the proton direction), this dependence does not occur for C. The protonation sites that result in the smallest barrier reduction for the pyrimidines is O4 for U (∼40 kJ mol−1 reduction, Δacid, Table 2), and the exocyclic amino group for C (∼50 kJ mol−1 reduction, Δacid, Table 2). For the purines, N7 protonation results in the largest barrier reduction (∼70 kJ mol−1), while protonation of the exocyclic amino group results in the smallest change in barrier (31.8−33.3 kJ mol−1). When N3 of A or G is protonated, the barrier is reduced by ∼40 kJ mol−1. The relative reaction energies of the acid-catalyzed pathways are similar to the uncatalyzed pathways, with the reaction energies being ∼80−190 kJ mol−1 and falling only ∼2−20 kJ mol−1 below the barriers. This indicates that protonation reduces the deglycosylation barriers and reaction energies to the same extent. However, acid catalysis does not provide the same stabilization to the PC as base catalysis, which leads to reaction energies of ∼50 to 80 kJ mol−1 for HCOO−···H2O or ∼ −40 to −60 kJ mol−1 for OH−. Therefore, a negatively charged species (e.g., OH− or HCOO−) is necessary to activate the water nucleophile and neutralize the positive charge forming in the TS, and thereby help stabilize the PC. Our calculated barrier heights for the most stable acidcatalyzed pathways for A and G hydrolysis are in near quantitative agreement with experimental data.70 Specifically, the experimentally determined acid-catalyzed RNA hydrolysis

Figure 6. Selected IEF-PCM-B3LYP/6-31G(d) bond lengths (Å) and angles (degrees in parentheses) in reactant complexes (RC), transition state (TS), and product complexes (PC) for the lowest barrier pathways of the acid-catalyzed deglycosylation of adenine (A−N7H), cytosine (C−O2H(N1)), guanine (G−N7H), and uracil (U−O2H(N3)) containing RNA nucleosides facilitated by the unactivated H2O nucleophile.

representative examples. Specifically, these pathways include those involving protonation at N7 of A or G and O2 of C or U. Structural parameters for all reaction pathways can be found in Tables S6−S9. We note that the orientation of the added proton can vary depending on the heteroatoms neighboring the nucleobase acceptor site, with the orientation defined in brackets. For example, the proton at O2 of C can be directed either toward N1 (denoted as C−O2H(N1)) or N3 (denoted H

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The Journal of Physical Chemistry B barriers for A and G are 111.7 and 111.3 kJ mol−1, respectively, which closely match our corresponding calculated barriers of 119.6 and 126.4 kJ mol−1, respectively. Our slightly higher barriers could likely be improved by modeling the reaction in explicit solvent, since the water nucleophile can only transfer a proton to 2′-OH in our model, while transfer to the bulk solvent would likely be more beneficial. Additionally, explicit solvation by even a few water molecules could stabilize the charge separated species and thereby further reduce the barrier, with up to four discrete water molecules having been previously shown to lower the barrier for the deglycosylation of the natural DNA nucleosides.31,32 More importantly, the nucleobase protonation sites that result in the greatest barrier reductions match sites that many enzymes are proposed to target. For example, π−π stacking between aromatic amino acids and the nucleobase has been proposed to facilitate solvent-mediated protonation of N7 of the purines by IAG-NH.22 This proposal correlates with our results that N7 protonation leads to the greatest barrier reduction for deglycosylation of RNA purines. Furthermore, protonation of O2 of C or U has been proposed as part of the activation mechanisms of CU-NH26 and RihC,85 which correlates with our findings that O2 is the most favorable activation site for RNA pyrimidines. Thus, our models correctly predict the effects of acid catalysis reported in the experimental literature, and provide valuable insights into nucleobase protonation sites that have the greatest catalytic impact, which rationalizes why enzymes tend to target specific nucleobase acceptor sites. Combined Effects of Acid and Base Catalysis. In general, the acid-catalyzed pathways afford greater barrier reductions than the base-catalyzed pathways (i.e., Δacid > Δbase, Table 2). Specifically, acid catalysis results in an up to 82.5 kJ mol−1 reduction over the uncatalyzed pathway, while base catalysis results in an up to 62.5 kJ mol−1 (OH−) or 18.3 kJ mol−1 (HCOO−···H2O) reduction. Nevertheless, the differences between the acid- and base-catalyzed pathways can be minimal, especially for strong base catalysis (i.e., the OH− nucleophile). Additionally, the reaction energies for the acid-catalyzed pathways are near equivalent to the barriers, suggesting the presence of a negatively charged species (e.g., HCOO− or OH−) is essential for favorable reaction thermodynamics. Therefore, both acid and base catalysis may significantly impact the deglycosylation reaction. Indeed, the active sites of enzymes that catalyze nucleic acid deglycosylation typically contain both acidic and basic residues that can facilitate the reaction.14−18,22−25,45−59 To examine the simultaneous impact of both acid and base catalysis, we modeled the departure of protonated nucleobases assisted by either OH− or HCOO−··· H2O. The most significant barrier reductions relative to uncatalyzed RNA hydrolysis result when both acid and base catalysis are employed, which leads to an up to ∼120 kJ mol−1 decrease in the barrier when OH− is the nucleophile or ∼100 kJ mol−1 when HCOO−···H2O is the nucleophile (Δacid−base, Table 2). Regardless, a comparison of the acid-catalyzed pathways for the OH− or HCOO−···H2O nucleophile to the corresponding acidcatalyzed pathways for the unactivated water nucleophile reveals that the presence of the base does not change the protonation site that results in the greatest barrier reduction. Specifically, acid catalysis directed at N7 of the purines or O2 of the pyrimidines results in the lowest excision barriers regardless of the nucleophile. Furthermore, regardless of the presence of acid catalysis, the barriers decrease and the reactions become

increasingly exothermic with stronger nucleophile activation (i.e., the barriers and reaction energies decrease according to the nucleophile as H2O > HCOO−···H2O > OH−, Table S14). While the combined efforts of acid and base catalysis yields the lowest barriers, it is of interest to determine whether the effects of acid and base catalysis on the deglycosylation barriers are additive, less than additive, or synergistic. Therefore, Table 2 (Δsum − Δacid−base) compares the sum of the individual acid and base contributions (Δsum = Δacid + Δbase) to the calculated effects of the simultaneous presence of acid and base catalysis (Δacid−base, Table 2). Whether the combined effects of acid and base catalysis are additive, less than additive, or synergistic depends on the level of nucleophile activation through base catalysis, the nucleobase, and the nucleobase protonation site. For example, when OH− is the nucleophile, Δacid−base ≈ Δsum (less than ∼10 kJ mol−1 difference, Table 2) for each purine deglycosylation pathway, indicating that the effects of acid and base catalysis are additive. Similarly, for the HCOO−···H2O deglycosylation pathways, Δsum ≈ Δacid−base in most pathways regardless of the identity of the nucleobase (Table 2). Conversely, the effects of acid and base catalysis are significantly diminished when simultaneously present for the C or U hydrolysis pathways facilitated by OH−, with Δacid−base being up to ∼30 kJ mol−1 less than Δsum. Finally, the G−N3H and U−O2H(N1) pathways exhibit significant acid−base synergy when HCOO−···H2O is the nucleophile, with Δacid−base being 20 kJ mol−1 larger than Δsum. Similar to the barriers, the effects of combined acid−base catalysis on the reaction energies are varied (Table S14), with the effects generally being additive (Δsum − Δacid−base < 10 kJ mol−1) but being synergistic (by up to ∼35 kJ mol−1) in some instances. Regardless of the additive nature of acid−base catalysis, our data emphasizes that both catalytic approaches are crucial for reducing otherwise prohibitive RNA nucleoside hydrolysis barriers, and both strategies likely play an important role in enzymatic systems.

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CONCLUSION The current study used several computational models to examine RNA nucleoside hydrolytic deglycosylation. First, a comparison of RNA and DNA hydrolysis facilitated by the HCOO−···H2O nucleophile revealed the 2′-hydroxy group in RNA nucleosides imparts stability to the glycosidic bond, which agrees with experimental results for purine deglycosylation under acidic conditions,70 and extends the conclusion to the pyrimidines and basic environments. Second, the effects of base catalysis were evaluated by considering hydrolysis mediated by an OH− nucleophile, which leads to significantly lower barriers than unactivated or HCOO− activated hydrolysis, as well as exothermic reactions. Third, the effect of acid catalysis was considered by protonation of nucleobase hydrogen-bond acceptor sites. Interestingly, the nucleobase protonation sites that result in the greatest barrier reductions are the same sites typically targeted by enzymes that catalyze RNA deglycosylation reactions. However, although acid reduces the hydrolysis barriers, the corresponding product complexes are only slightly more stable than the TS, implying that both acid and base catalysis is important. Indeed, simultaneously invoking both acid and base catalytic approaches is the most effective strategy for reducing RNA deglycosylation barriers. Whether the effect of concomitant acid and base catalysis is additive, less than additive, or synergistic compared to the sum of the individual effects is dependent on the nucleobase, protonation site, and nucleophile. Overall, this work establishes a fundamental I

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understanding of the intrinsic stability of the RNA glycosidic bond and the effects of acid and base catalysis on RNA deglycosylation. Our findings provide important mechanistic details for scientists studying enzyme-mediated RNA deglycosylation reactions that can be used as a basis for future largescale modeling of enzymatic reactions and to design TS analogues that inhibit RNA-targeting enzymes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b09620. Reaction pathways for the HCOO−···H2O catalyzed pathways without the presence of a bridging water (Figure S1) and selected bond lengths, angles, and dihedral angles for each pathway examined (Tables S1− S13) and full citation for refs 9, 78, and 82 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (403) 329-2323. Fax: (403) 329-2057. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada (NSERC, 249598-07), Canada Research Chairs Program (950-228175), and Canada Foundation for Innovation (22770) for financial support. S.A.P.L. acknowledges NSERC (CGS-D), and S.A.P.L. and J.D.K. acknowledge the University of Lethbridge for student scholarships. Computational resources from the Upscale and Robust Abacus for Chemistry in Lethbridge (URACIL) and those provided by Westgrid and Compute/Calcul Canada are greatly appreciated.

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ABBREVIATIONS A, adenine; AAG, alkyladenine DNA glycosylase; C, cytosine; CU-NH, cytidine-uridine nucleoside hydrolase; DNA, deoxyribonucleic acid; G, guanine; IAG-NH, inosine-adenosineguanosine nucleoside hydrolase; IU-NH, inosine-uridine nucleoside hydrolase; MutY, adenine DNA glycosylase; NH, nucleoside hydrolase; PC, product complexes; RihC, inosineuridine nucleoside hydrolase; RC, reactant complexes; rxn, reaction energy; RNA, ribonucleic acid; TS, transition state complexes; U, uracil; UDG, uracil DNA glycosylase

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REFERENCES

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DOI: 10.1021/acs.jpcb.6b09620 J. Phys. Chem. B XXXX, XXX, XXX−XXX